Major Article Sodium Nitroprusside Has Leishmanicidal Activity

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Revista da Sociedade Brasileira de Medicina Tropical 49(1):68-73, Jan-Feb, 2016 Major Article http://dx.doi.org/10.1590/0037-8682-0266-2015

Sodium nitroprusside has leishmanicidal activity independent of iNOS

Natália Yoshie Kawakami[1], Fernanda Tomiotto-Pellissier[1], Allan Henrique Depieri Cataneo[1], Tatiane Marcusso Orsini[1], Ana Paula Fortes Dos Santos Thomazelli[1], Carolina Panis[2], Ivete Conchon-Costa[1] and Wander Rogério Pavanelli[1]

[1]. Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina, Paraná, Brasil. [2]. Laboratório de Mediadores Inﬂ amatórios, Universidade Estadual do Oeste do Paraná, Francisco Beltrão, Paraná, Brasil.

ABSTRACT Introduction: Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania. Cutaneous leishmaniasis is the most common form, with millions of new cases worldwide each year. Treatments are ineffective due to the toxicity of existing drugs and the resistance acquired by certain strains of the parasite. Methods: We evaluated the activity of sodium nitroprusside in macrophages infected with Leishmania (Leishmania) amazonensis. Phagocytic and microbicidal activity were evaluated by phagocytosis assay and promastigote recovery, respectively, while cytokine production and nitrite levels were determined by ELISA and by the Griess method. Levels of iNOS and 3-nitrotyrosine were measured by immunocytochemistry. Results: Sodium nitroprusside exhibited in vitro antileishmanial activity at both concentrations tested, reducing the number of amastigotes and recovered promastigotes in macrophages infected with L. amazonensis. At 1.5µg/mL, sodium nitroprusside stimulated levels of TNF-α and nitric oxide, but not IFN-γ. The compound also increased levels of 3-nitrotyrosine, but not expression of iNOS, suggesting that the drug acts as an exogenous source of nitric oxide. Conclusions: Sodium nitroprusside enhances microbicidal activity in Leishmania-infected macrophages by boosting nitric oxide and 3-nitrotyrosine. Keywords: Leishmaniasis. Nitric oxide. 3-nitrotyrosine. Leishmania amazonensis. Sodium nitroprusside.

In addition, some strains of the parasite have acquired resistance INTRODUCTION to these drugs(5). Macrophages mount various mechanisms to combat Leishmaniasis, a group of infectious diseases found parasites, including oxidative burst, acidiﬁ cation of vesicles, worldwide, is caused by protozoa of the genus Leishmania. and expression of inducible nitric oxide synthase (iNOS)(6). Approximately 20 to 40 thousand deaths each year are attributed iNOS synthesizes nitric oxide, a highly reactive, membrane- to this disease, which can manifest in various forms with diffusible molecule used to control various pathogens(7). Nitric different symptoms depending on the infecting species and oxide reacts with reactive oxygen species to generate reactive the host immune response. The main forms of the disease are nitrogen species such as peroxynitrite(8), which damage DNA, cutaneous, mucocutaneous, and visceral, of which the cutaneous inhibit enzymes, and peroxidize lipids(9). form is the most common, with 0.7 to 1.2 million new cases Leishmania spp. has evolved several mechanisms to evade each year in 98 countries(1). Pentavalent antimonials are the macrophage activity. For instance, parasites suppress nitric standard treatments, of which meglumine antimoniate and oxide production by taking up L-arginine, a required substrate sodium stibogluconate are the most frequently used(2). However, for iNOS, as well as by inhibiting the release of IFN-γ and these drugs require long treatment regimens and parenteral (3) TNF-α, proinﬂ ammatory cytokines that stimulate expression or intralesional administration , and cause numerous side (10) effects, including pancreatitis, hepatitis, and cardiotoxicity(3) (4). of this enzyme . These observations suggest that exogenous sources of nitric oxide may potentially be used to control leishmaniasis. Indeed, studies have shown that such sources are active against Leishmania in vitro(11) and in vivo(12), as well as other Corresponding author: Dr. Wander Rogério Pavanelli. (13) e-mail: [email protected] I [email protected] microorganisms, including Strongyloides venezuelensis , (14) (15) Received 25 August 2015 Paracoccidioides brasiliensis , and Trypanosoma cruzi . One Accepted 4 December 2015 such source is sodium nitroprusside (Na2[Fe(CN)5NO]•2H2O),

68 Kawakami NY et al. - Leishmanicidal activity of sodium nitroprusside an inorganic compound(16) active against promastigotes and Nitrite levels axenic amastigotes of Leishmania (Leishmania) amazonensis(17). Nitrite was also determined in the supernatant of cultures In this report, we demonstrate that sodium nitroprusside enhances the microbicidal activity of Leishmania-infected of infected macrophages treated with sodium nitroprusside. macrophages by enhancing production of nitric oxide and Nitric oxide was measured with the Griess reagent according (19) 3-nitrotyrosine. to published methods . Brieﬂ y, aliquots were diluted in 45g/L glycine pH 9.7, and treated for 10 min with cadmium granules

previously activated with 5mM CuSO4. Subsequently, 200µL METHODS of this mixture reacted for 10 min at room temperature with an equal volume of Griess reagent. Tubes were then centrifuged Leishmania (Leishmania) amazonensis at 10,845 ×g for 2 min at 25°C, and transferred to 96-well Promastigotes of L. amazonensis (MHOM/BR/1989/166MJO) microplates in triplicate. Absorbance at 550nm was determined were maintained in 199 medium pH 7.18-7.22 (Invitrogen-GIBCO) in a microplate reader. A calibration curve was constructed using supplemented with 10% fetal bovine serum (Invitrogen-GIBCO), dilutions of NaNO2. 10mM HEPES, 0.1% human urine, 0.1% L-glutamine, 10U/mL penicillin, 10µg/mL streptomycin (Invitrogen-GIBCO), and 10% Immunocytochemistry for iNOS and 3-nitrotyrosine sodium bicarbonate. Cultures were grown in 25cm2 ﬂ asks at 24°C. Slides with adherent macrophages were prepared in triplicate as described for the phagocytic assay, and labeled Culture of peritoneal macrophages and by the streptavidin-biotin method (Universal Dako LSAB®+ phagocytosis assay System-HRP Kit, DAKO Japan, Kyoto, Japan) without Macrophages were obtained from the peritoneal cavity microwave pretreatment. Slides were then treated with of BALB/c mice, re-suspended in RPMI 1640 medium pH 10% Triton-X for 15 min, washed with phosphate-buffered

7.2 (Gibco BRL), and incubated for 2h at 37ºC and 5% CO2 saline, and incubated in 1% fetal bovine serum for 30 min. in 24-well plates (5 × 105 cells per well) with 13mm glass Subsequently, slides were probed overnight at 4 ºC with 1:300 coverslips with 200µL RPMI 1640 medium. Adherent cells dilutions of rabbit polyclonal primary antibodies against iNOS were infected for 2h with L. amazonensis promastigotes at a and 3-nitrotyrosine (Santa Cruz Biotechnology), and then ratio of 1:5, washed with phosphate-buffered saline to remove with biotinylated anti-rabbit, anti-mouse, and anti-goat IgG non-phagocytized parasites, and treated for 24h at 37ºC and (LSAB+ System-HRP, DAKO, Japan, Kyoto, Japan) for 2h at 5% CO2 with RPMI 1640 (control) or with 0.5 and 1.5µg/mL room temperature. Negative controls were performed omitting sodium nitroprusside. Subsequently, cells were stained with the primary antibodies. Horseradish peroxidase activity was Giemsa, and 200 cells per sample were imaged at 1,000× under visualized with H2O2 and 3,3'-diaminobenzidine for 5 min, and a CX31RBSFA light microscope (Olympus Optical Co. Ltd., cells were counterstained with Harris hematoxylin (Merck). Tokyo, Japan) to quantify the number of infected macrophages Finally, slides were digitally imaged in color at 400× using a and the average number of amastigotes per macrophage. BX41 photomicroscope (Olympus Optical Co. Ltd., Tokyo, Japan). Representative fi elds of view from 10 images of each replicate Promastigote recovery were scored semi-quantitatively using color deconvolution in ImageJ (NIH, USA). Pixels with intensity 0-255 were Promastigote recovery was performed as previously categorized as strongly positive (3+, intensity 0-60), positive described(18). Briefl y, peritoneal macrophages (5 × 105 cells) (2+, intensity 61-120), weakly positive (1+, intensity were infected with L. amazonensis, treated for 24h at 37ºC and 121-170), and negative (0, intensity 171-230), as previously 5 % CO with 0.5 and 1.5µg/mL sodium nitroprusside, washed 2 described(20). Slides that were not probed with primary antibody with phosphate-buffered saline, and incubated at 24°C with 199 were used as negative control. medium to induce differentiation of intracellular amastigotes into promastigotes. Promastigotes recovered were counted daily in a Neubauer chamber for three days after infection. Statistical analysis Data are reported as mean ± standard error of the mean. Cytokine levels Duplicate datasets from three independent experiments with Supernatants were collected from cultures of infected three animals per experiment were analyzed in Prism GraphPad macrophages 24h after treatment with sodium nitroprusside, 5.00 (GraphPad Software, Inc., USA). Data were found to centrifuged at 460 ×g for 7 min at 4°C, and stored at -80°C be normally distributed by Kolmogorov-Smirnov test, and until analysis. TNF-α, IFN-γ, and IL-12-p70 were determined variances were found to be homogeneous by F test. Treatments by enzyme-linked immunosorbent assay (ELISA) according to were compared by Student’s t-test or analysis of variance the manufacturer's instructions (eBiosciences®, USA). Plates followed by Tukey’s test for multiple comparisons. Differences were read at 450nm using a plate reader (Thermo-TP-Reader). were considered statistically signifi cant when p < 0.05.

69 Rev Soc Bras Med Trop 49(1):68-73, Jan-Feb, 2016

Ethics statement with different concentrations of the compound for 24h after infection. We found that exposure to sodium nitroprusside for 24h Female BALB/c mice weighing approximately 25-30g did not signifi cantly affect the number of infected macrophages. and aged 6-8 weeks were housed in pathogen-free conditions However, the number of amastigotes per macrophage was according to protocols approved by the Institutional Animal Care signifi cantly reduced in cells treated with 0.5µg/mL (p = 0.0188) and Use Committee at Londrina State University. This study was and 1.5µg/mL (p = 0.0409) sodium nitroprusside (Figure 1A approved by the Londrina State University Ethics Committee and 1B). Accordingly, sodium nitroprusside also reduced the for Animal Experimentation (33064.2012.42). number of promastigotes recovered, with p < 0.0001 for both concentrations 72h after exposure (Figure 1C). The data indicate RESULTS that treatment with sodium nitroprusside for 24h enhanced leishmanicidal activity in macrophages. Sodium nitroprusside alters phagocytic capacity Sodium nitroprusside increases nitric oxide and and increases microbicidal activity favors formation of 3-nitrotyrosine To characterize the impact of sodium nitroprusside on Exposure to 1.5µg/mL sodium nitroprusside for 24h phagocytic and microbicidal activity, macrophages were treated signifi cantly increased nitric oxide (p = 0.0304, Figure 2A)

A 200 4 B

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Promastigotes (x10 0.1 ** *** * 0.0 0 24 48 72 Time (hours)

FIGURE 1 – Peritoneal macrophages from BALB/c mice were infected in vitro with Leishmania amazonensis and treated for 24h with 0.5 and 1.5μg/mL sodium nitroprusside (SNP). A) Number of infected macrophages. B) Number of amastigotes per macrophage. Dashed line indicates the number of (A) infected macrophages and (B) the amount of internalized parasites after infection period (2 h). C) Leishmania amazonensis promastigotes recovered over three days after infection. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control, by one-way ANOVA followed by Tukey test.

70 Kawakami NY et al. - Leishmanicidal activity of sodium nitroprusside in macrophages infected with L. amazonensis. Accordingly, sodium nitroprusside remains in use due to its effectiveness and there was increased immunostaining for 3-nitrotyrosine rapid action(26) (28), despite reports of cyanide toxicity. (p = 0.0467, Figure 2B), implying that sodium nitroprusside acts A previous study in vitro demonstrated that sodium as an exogenous source of nitric oxide. Notably, 3-nitrotyrosine nitroprusside decreased the number of L. amazonensis colocalized with the parasite in some cells. promastigotes and axenic amastigotes in a dose-dependent manner(17). In accordance with this result, we observed that Sodium nitroprusside does not induce iNOS expression the drug reduced the number of intracellular amastigotes, and, consequently, the number of promastigotes recovered Semi-quantitative immunocytochemistry demonstrated that (Figure 1B and 1C). Thus, we investigated the mechanism by sodium nitroprusside exposure was not associated with elevated which sodium nitroprusside enhanced leishmanicidal activity. expression of iNOS (Figure 2B), indicating that the increase in We found that nitric oxide levels increased in the supernatant nitric oxide was exogenous. of cultured macrophages exposed to 1.5µg/mL sodium nitroprusside (Figure 2A). Consequently, 3-nitrotyrosine was generated, indicating enhanced formation of reactive nitrogen Sodium nitroprusside increases TNF-α radicals(29). Notably, 3-nitrotyrosine and nitrated proteins Treatment of infected macrophages for 24h with 1.5µg/mL accumulated in phagosomes and in intracellular parasites sodium nitroprusside increased TNF-α (p = 0.0065, Figure 3A), (Figure 2B), reinforcing the idea that parasite clearance depends but not IFN-γ and IL-12 (Figure 3B and 3C). on reactive nitrogen species. Indeed, 3-nitrotyrosine peaks early in infection in leishmaniasis-resistant C57BL6 mice, presaging DISCUSSION a subsequent decline in parasitosis. In contrast, 3-nitrotyrosine peaks late in leishmaniasis-susceptible BALB/c mice(30), Nitric oxide is well known to be a key effector in clearing implying that 3-nitrotyrosine formation is a relevant indicator Leishmania(21), although the parasite is capable of suppressing of antiparasitic activity. nitric oxide production via several mechanisms(22) (23). Thus, In addition to 3-nitrotyrosine, the outcome of Leishmania drugs that release nitric oxide, including sodium nitroprusside(24), infection is critically dependent on the host immune response. may enhance leishmanicidal activity in macrophages. The In mice, protective immunity depends on the ability of IL-12 pharmacological characteristics of these drugs were established in to trigger Th1 activity and release of IFN-γ(22) (31) (32). Indeed, 1955(25), and clinical application has since widened(26) (27). Indeed, macrophages stimulated with IL-12 secrete IFN-γ(33), although

iNOS A 3-nitrotirosine B 200 4

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g/mL g/mL g/mL ntro g/mL μ μ μ μ Control Co Contro .5 .5 .5 1.5 P 0 P 0 SN SNP SN SNP 1

FIGURE 2 - A) Nitrite levels in peritoneal macrophages isolated from BALB/c mice, infected in vitro with Leishmania amazonensis, and treated for 24h with 0.5 and 1.5μg/mL sodium nitroprusside (SNP). B) iNOs and 3-nitrotyorsine, as measured by immunocytochemistry, in BALB/c peritoneal macrophages infected with L. amazonensis, and treated with 1.5µg/mL sodium nitroprusside for 24h. Pixel intensities (ICC score) were scored as strongly positive (3+), positive (2+), weakly positive (1+), and negative (0). Sodium nitroprusside did not affect expression of iNOS, but increased 3-nitrotyrosine, which colocalized with the parasite (arrow). *, p < 0.05 vs. control, by t test or one-way ANOVA followed by Tukey test.

71 Rev Soc Bras Med Trop 49(1):68-73, Jan-Feb, 2016

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FIGURE 3 - TNF-α (A), IL-12 (B), and IFN-γ (C), as measured by ELISA, in BALB/c peritoneal macrophages infected in vitro with Leishmania amazonensis and treated with 0.5 and 1.5μg/mL sodium nitroprusside (SNP) for 24h. *p < 0.01 vs. control, by one-way ANOVA followed by Tukey test.

this cytokine is primarily produced by natural killer cells, CD4+, CONFLICT OF INTEREST and CD8+ T lymphocytes(34). We note that Leishmania is capable of suppressing IL-12 expression(35) (36). Importantly, we found that IL-12 production (Figure 3B) and IFN-γ secretion were The authors declare that there is no confl ict of interest. not affected by sodium nitroprusside (Figure 3C). In contrast, sodium nitroprusside stimulated levels of TNF-α REFERENCES (Figure 3A), a key cytokine involved in macrophage expression of iNOS(37). However, the increase in TNF-α did not stimulate 1. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. iNOS expression (Figure 2B), in line with published data WHO Leishmaniasis Contro Team. Leishmaniasis worldwide and demonstrating that exogenous sources of nitric oxide suppress global estimates of its incidence. PLoS One 2012; 7:e35671. expression of this enzyme(38) (39). Thus, we conclude that the 2. Palumbo E. Treatment stra tegies for mucocutaneous leishmaniasis. antileishmanial activity of sodium nitroprusside depends on its J Glob Infect Dis 2010; 2:147-150. properties as a source of nitric oxide(40) (41). 3. Frézard F, Demicheli C. New delivery strategies for the old pentavalent antimonial drugs. Expert Opin Drug Deliv 2010; In summary, we have demonstrated in vitro that sodium 7:1343-1358. nitroprusside enhances leishmanicidal activity in macrophages 4. Sundar S, Chakravarty J. Leishmaniasis: an update of current infected with L. amazonensis via release of nitric oxide. pharmacotherapy. Expert Opin Pharmacother 2013; 14:53-63. Even though the drug has some toxicity and is challenging to 5. Kaur G, Rajput B. Comparative analysis of the omics technologies administer, the results provide a rationale for further studies used to study antimonial, amphotericin B, and pentamidine in vivo, in light of the serious limitations of current therapies, resistance in leishmania. J Parasitol Res 2014; 2014:726328. which have limited effi cacy and signifi cant toxicity, and require 6. Cunningham AC. Parasitic adaptive mechanisms in infection by long treatment regimens. leishmania. Exp Mol Pathol 2002; 72:132-141. 7. Qadoumi M, Becker I, Donhauser N, Röllinghoff M, Bogdan C. ACKNOWLEDGMENTS Expression of inducible nitric oxide synthase in skin lesions of patients with american cutaneous leishmaniasis. Infect Immun 2002; 70:4638-4642. Dr. A. Leyva helped with English translation and editing. 8. Rosselli M, Keller PJ, Dubey RK. Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Hum Reprod Update 1998; 4:3-24. FINANCIAL SUPPORT 9. James SL. Role of nitric oxide in parasitic infections. Microbiol Rev 1995; 59:533-547. This work was financially supported by Secretaria de 10. Perrella-Balestieri FM, Queiroz AR, Scavone C, Costa VM, Barral- Ciencia e Tecnologia do Paraná, Conselho Nacional de Netto M, Abrahamsohn IA. Leishmania (L.) amazonensis-induced Desenvolvimento e Pesquisa (CNPq: 82195/2013-4), and State inhibition of nitric oxide synthesis in host macrophages. Microbes University of Londrina. Infect 2002; 4:23-29.

72 Kawakami NY et al. - Leishmanicidal activity of sodium nitroprusside

11. Tfouni E, Truzzi DR, Tavares A, Gomes AJ, Figueiredo LE, Franco DW. 25. Page IH, Corcoran AC, Dustan HP, Koppanyi T. Cardiovascular Biological activity of ruthenium nitrosyl complexes. Nitric Oxide actions of sodium nitroprusside in animals and hypertensive 2012; 26:38-53. patients. Circulation 1955; 11:188-198. 12. Miranda MM, Panis C, Cataneo AH, da Silva SS, Kawakami NY, 26. Hottinger DG, Beebe DS, Kozhimannil T, Prielipp RC, Belani Lopes LG, et al. Nitric oxide and Brazilian propolis combined KG. Sodium nitroprusside in 2014: A clinical concepts review. accelerates tissue repair by modulating cell migration, cytokine J Anaesthesiol Clin Pharmacol 2014; 30:462-471. production and collagen deposition in experimental leishmaniasis. 27. Taylor TH, Styles M, Lamming AJ. Sodium nitroprusside as a PLoS One 2015; 10:e0125101. hypotensive agent in general anaesthesia. Br J Anaesth 1970; 13. Ruano AL, López-Abán J, Fernández-Soto P, de Melo AL, Muro A. 42:859-864. Treatment with nitric oxide donors diminishes hyperinfection by 28. Friederich JA, Butterworth JF. Sodium nitroprusside: twenty years Strongyloides venezuelensis in mice treated with dexamethasone. and counting. Anesth Analg 1995; 81:152-162. Acta Trop 2015; 152:90-95. 29. Schopfer FJ, Baker PR, Freeman BA. NO-dependent protein 14. Pavanelli W, da Silva JJ, Panis C, Cunha TM, Costa IC, de Menezes nitration: a cell signaling event or an oxidative inﬂ ammatory MC, et al. Experimental Chemotherapy in Paracoccidioidomycosis response? Trends Biochem Sci 2003; 28:646-654. Using Ruthenium NO Donor. Mycopathol 2011; 172:95-107. 30. Linares E, Giorgio S, Mortara RA, Santos CX, Yamada AT, Augusto 15. Silva JJN, Guedes PMM, Zottis A, Balliano TL, Nascimento- O. Role of peroxynitrite in macrophage microbicidal mechanisms Silva FO, França Lopes LG, et al. Novel ruthenium complexes as in vivo revealed by protein nitration and hydroxylation. Free Radic potential drugs for Chagas's disease: enzyme inhibition and in vitro/ Biol Med 2001; 30:1234-1242. in vivo trypanocidal activity. Br J Pharmacol 2010; 160:260-269. 31. Heinzel FP, Rerko RM, Hatam F, Locksley RM. IL-2 is necessary 16. Bates JN, Baker MT, Guerra Jr R, Harrison DG. Nitric oxide for the progression of leishmaniasis in susceptible murine hosts. J Immunol 1993; 150:3924-3931. generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required. 32. Sypek JP, Chung CL, Mayor SE, Subramanyam JM, Goldman Biochem Pharmacol 1991; 42:S157-S165. SJ, Sieburth DS, et al. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune 17. Genestra M, Soares-Bezerra RJ, Gomes-Silva L, Fabrino DL, Bellato- response. J Exp Med 1993; 177:1797-1802. Santos T, Castro-Pinto DB, et al. In vitro sodium nitroprusside-

mediated toxicity towards Leishmania amazonensis promastigotes 33. Gessani S, BelardellI F. IFN-gamma expression in macrophages and axenic amastigotes. Cell Biochem Funct 2008; 26:709-717. and its possible biological signiﬁ cance. Cytokine Growth Factor Rev 1998; 9:117-123. 18. Da Silva SS, Thomé GS, Cataneo AHD, Miranda MM, Felipe

I, Guadalupe C, et al. Brazilian propolis antileishmanial and 34. Farrar MA, Schreiber RD. The molecular cell biology of interferon- immunomodulatory effects. Evid Based Complement Alternat Med gamma and its receptor. Annu Rev Immunol 1993; 11:571-611. 2013; 2013:673058. 35. Lapara NJ, Kelly BL. Suppression of LPS-induced inﬂ ammatory responses in macrophages infected with Leishmania. J Inﬂ amm 19. Panis C, Mazzuco TL, Costa CZ, Victorino VJ, Tatakihara VL, (Lond) 2010; 7:8. Yamauchi LM, et al. Trypanosoma cruzi: Effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, 36. Murphy TL, Cleveland MG, Kulesza P, Magram J, Murphy KM. nitric oxide and iNOS expression in cardiac tissue in the acute phase Regulation of interleukin 12 p40 expression through an NF-kappa of infection in mice. Exp Parasitol 2011; 127:58-65. B half-site. Mol Cell Biol 1995; 15:5258-5267.

20. Chatterjee S, Malhotra R, Varghese F, Bukhari AB, Patil A, 37. Fonseca SG, Romão PR, Figueiredo F, Morais RH, Lima HC, Budrukkar A, et al. Quantitative Immunohistochemical Analysis Ferreira SH, et al. TNF-alpha mediates the induction of nitric oxide Reveals Association between Sodium Iodide Symporter and synthase in macrophages but not in neutrophils in experimental Estrogen Receptor Expression in Breast Cancer. PLoS ONE 2013; cutaneous leishmaniasis. Eur J Immunol 2003; 33:2297-3306. 8:e54055. 38. Chang K, Lee SJ, Cheong I, Billiar TR, Chung HT, Han JA, et al. Nitric oxide suppresses inducible nitric oxide synthase expression 21. Horta MF, Mendes BP, Roma EH, Noronha FSM, Macêdo JP, by inhibiting post-translational modiﬁ cation of IkappaB. Exp Mol Oliveira LS, et al. Reactive oxygen species and nitric oxide in Med 2004; 36:311-324. cutaneous leishmaniasis. J Parasitol Res 2012; 2012:203818.

39. Colasanti M, Persichini T, Menegazzi M, Mariotto S, Giordano 22. Isnard A, Shio MT, Olivier M. Impact of Leishmania metalloprotease E, Calderena CM, et al. Induction of nitric oxide synthase mRNA GP63 on macrophage signaling. Front Cell Infect Microbiol 2012; expression. Suppression by exogenous nitric oxide. J Biol Chem 2:72. 1995; 270:26731-26733.

23. Shio MT, Hassani K, Isnard A, Ralph B, Contreras I, Gomez MA, 40. Emre A, Bayram O, Salman B, Ercan S, Anadol Z, Akin O. Sodium et al. Host cell signalling and leishmania mechanisms of evasion. Nitroprusside as a Nitric Oxide Donor in a Rat Intestinal Ischemia- J Trop Med 2012; 2012:819512. Reperfusion Model. Clinics 2008; 63:91-96. 24. Lockwood A, Patka J, Raninovich M, Wyatt K, Abraham P. Sodium 41. William M, Vien J, Hamilton E, Garcia A, Bundgaard H, Clarke nitroprusside-associated cyanide toxicity in adult patients - fact or RJ, et al. The nitric oxide donor sodium nitroprusside stimulates the ﬁ ction? A critical review of the evidence and clinical relevance. Na+–K+ pump in isolated rabbit cardiac myocytes. Journal Physiol J Clin Trials - Dovepress 2010; 2:133-148. 2005; 565:815-825.